Project Summary Middle East Respiratory Syndrome coronavirus (MERS-CoV/MERS) is a recently emerged zoonotic pathogen that has caused almost 1,800 cases and over 600 deaths since its 2012 discovery. MERS is the second virulent coronavirus to emerge in the last fifteen years, following SARS-CoV (SARS) in 2003, further demonstrating that coronaviruses are a continuing threat to global public health. While MERS, SARS, and the murine coronaviruses all belong to the same genus, Betacoronavirus, MERS is highly divergent from both and its genome encodes a novel set of accessory proteins. All viruses must manipulate diverse arms of the host antiviral response to ensure replicative success and transmission, and coronavirus accessory proteins participate in these essential functions. Due to its novelty and divergence from previously studied coronaviruses, the MERS accessory proteins are largely uncharacterized and lack significant sequence homology with other known viral proteins. We have used structural modeling to identify the MERS NS4b accessory protein as a homolog of the mouse hepatitis virus (MHV) NS2 accessory protein, which was previously described by our lab as an antagonist of the antiviral enzyme RNase L. Both proteins are homologs of the LigT-like 2H-phosphoesterases (2H-PEs), a large family of prokaryotic and eukaryotic proteins characterized by 2 HxS/T catalytic motifs separated by 80-100 amino acids. NS2 is critical for MHV replication in myeloid cells and the mouse liver, and inactivation of NS4b enzymatic activity results in RNase L activation during MERS infection of human airway epithelial Calu-3 cells. Antagonism of RNase L by either MHV NS2 or MERS NS4b is dependent on 2?,5? phosphodiesterase activity mediated by the catalytic histidines. Despite the similarity between these two proteins, our lab has identified striking differences and significant preliminary data which, combined with reports in the literature that NS4b can antagonize expression from the interferon- ? promoter in reporter assays, suggest it has an additional function as compared to MHV NS2. Notably, whereas MHV NS2 is exclusively cytoplasmic, MERS NS4b contains a strong, functional NLS and localizes primarily to the nucleus. Our lab previously demonstrated that nuclear localization is completely dispensable for antagonizing RNase L, and the NS4b NLS is conserved in all known MERS-like viruses, strongly supporting the idea of a nuclear function. Additionally, whereas MHV NS2 cleaves only 2?-5? linked phosphodiester bonds, MERS NS4b cleaves both 2?-5? and 3?-5? linked bonds, giving it broader substrate range and the ability to process RNA, as some prokaryotic and eukaryotic 2H-PEs are known to do. Our preliminary data strongly suggest that NS4b post-transcriptionally processes host mRNAs associated with the antiviral response and cell death, doing so in the nucleus through its enzymatic activity. Therefore, MERS NS4b is likely the first known viral 2H-PE to act in the nucleus. This research may illuminate a new mechanism by which coronaviruses interact with the host, and determine whether NS4b is a plausible therapeutic target during MERS infection.